Roles and Actions of Kinases:
Receptor protein kinases coupled with complex downstream kinase and phosphatase mediated cascades and feedback loops play critical roles in signal transduction from the cell exterior into the nucleus where transcriptional regulation takes place. Upon receptor activation, this signal transduction is performed by the act of phosphate transfer to the serine, threonine, and tyrosine residues of proteins that results in enzyme activation or inactivation, changes in conformation, increased or decreased affinity for other proteins, appropriate localization, and in some cases targeting of proteins for degradation by the proteosome. Understandably, these processes are tightly regulated and balanced through control of external receptor ligands as well as expression of receptors, receptor antagonists, decoy receptors, and through redundancies or crosstalk between signaling pathways. Thus, it is accepted that kinases perform essential functions in regulating cell growth and proliferation, differentiation and cell development, cell division and aberrant mitogenesis. Kinases also mediate and regulate cell adhesion, angiogenesis, stress responses, cell-cell or cell-matrix interactions, and short range contact-mediated axional guidance. Mechanistically similar non-protein kinases, such as PI3Ks and SPK1, can also phosphorylate other ligands that contribute to the regulatory process (Brown J. R., BMC Evolutionary Biology (2011) 11(4): 1471-2148; Alvarez S. E., Nature (2010) 465: 1084-1088). Therefore, diseases and conditions where aberrant kinase activity plays a role are plentiful. However, the complexities of the systems biology combined with the structural homology of the kinase sites in the over 500 members of the human kinome presents a significant challenge for disease specific intervention by kinase inhibitors.
The Therapeutic Utility of Kinase Inhibitors:
With the advent of Imatinib (Deininger M., Blood (2005) 105 (7):2640-2653) the primary focus for kinase inhibitor development has been for the targeted treatment of specific cancers where mutation driven aberrant kinase activations are particularly significant. Applications for kinase inhibitors in cancer therapy continues to evolve and these utilities have been extensively reviewed (Zhang J., Nat. Rev. Cancer (2009) 9(1): 28-39). However, strong links exist between cancer progression and a pro-growth inflammatory environment have been established (Rakoff-Nahoum S., Yale J. Biol. Med. (2006), 79:123-130; Schmid M. C., Cancer Cell, (2011) 19(6): 715-727). In addition, a diverse set of kinases participate in chronic inflammatory diseases, such as rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, and chronic obstructive pulmonary disease, which are highly debilitating diseases that affect a large segment of our population. Moreover, it has become apparent that metabolic diseases such as type 2 diabetes, neuro-degenerative disorders such as Alzheimers, and cardiovascular diseases such as athlerosclerosis, also have a strong inflammatory component involving overactive kinase pathways. Therefore, selective inhibition of key kinases and their compensatory mechanisms continues to be pursued as a promising strategy for therapeutic intervention. (Garuti L., Current Medicinal Chemistry (2010) 17: 2804-2821).
Due to the complexity of signal transduction pathways, compensatory mechanisms often confound the initial therapeutic benefits seen with highly selective targeted kinase inhibitors. Conversely, undesired off-target effects can introduce significant toxicity. The ongoing challenge in the development of kinase inhibitors, particularly for chronic administration, is achieving the balance between efficacy and safety. Since, the aberrant activity of kinases is fundamental to many chronic diseases and cancers, much effort continues to be expended to understand their diverse and complex roles in basic physiology.
Kinases in Inflammatory Diseases:
Mitogen-activated protein (MAP) kinases are known to play key roles in the transmission of signals from cell surface receptors to transcription factors which up-regulate the expression of pro-inflammatory cytokines. The MAP kinase p38-α is participant in one pathway that regulates the production of the pro-inflammatory cytokines TNF-α, IL-1, IL-6 and IL-8, as well as the enzymes COX-2, MMP-1 and MMP-3. It has also been demonstrated that inhibiting p38-α kinase delays the onset of joint disease in animal models of arthritis (Mihara K., British Journal of Pharmacology (2008) 154:153-164) by arresting the over production of these pro-inflammatory cytokines (Schindler J. F. J. Dental Res. (2007), 86(9): 800-811). However, high hopes for p38 inhibitors as a single target therapy for chronic inflammation have not been realized in clinical studies which demonstrated these effects to be short lived, presumably by activation of compensatory mechanisms (Sweeney S. E., Nature Reviews Rheumatology (2009) 5: 475-477). However, recent reports of phase-II data in osteoarthritic patients using a sustained release formulations of p38 inhibitor, FX005, delivered intra-articularly to the knee look promising for both relief of pain and inflammation. More recently, redundant and non-redundant functions of the JNK isoforms JNK1 and JNK2 in the immune system and arthritis have been described (Guma M., Proc Natl Acad Sci USA. (2010), 107(51):22122-7; Hommes D., Gastroenterology. (2002), 122(1):7-14. Stambe C., Kidney Int (2003), 64:2121-2132; Ma F. Y., Laboratory Investigation (2009) 89: 470-484). Additionally, inhibitors of Janus family kinases (JAK1, JAK2, and JAK3) have demonstrated anti-inflammatory effects in animal models (Stump K L., Arthritis Research and Therapy (2011) 13:R68; Meyer D M., J. Inflammation (2010) 7(41):1-12). Consequently, interest in small-molecule therapeutics that target p38, JAK, and JNK isoforms for inflammatory diseases remains high (Liu C., J. Med. Chem. (2010) 53(18): 6629-6639).
Encouraging anti-inflammatory preclinical and clinical results with Imatinib, the well-known anti-cancer kinase inhibitor with Abl, PDGFR, c-KIT, and c-Raf activities, (Deininger M., Blood (2005) 105 (7):2640-2653.) has rekindled interest in the development of kinase inhibitors as anti-inflammatory agents (Iyoda M., Kidney International (2009), 75(10):1060-70; Ghofrani H. A., J Am Coll Cardiol (2009) 54:108-117; Louvet C, Proc. Natl. Acad. Sci. USA (2008) 105:18895-18900). Both protein and lipid kinases are now seen as potential targets for the attenuation of the inflammatory response. Macrophage colony stimulating factor receptor (CSF-1R or FMS) along with KIT, FLT3, and PDGFR-a/b, are members of the type-III receptor tyrosine kinase family which have enjoyed much attention as potential kinase targets (Tamura T. and Koch A., Anti-Inflamm Anti-Allergy Agents in Med. Chem. (2007) 6: 47-60). CSF-1R and its ligand (CSF-1) have been implicated in a range of macrophage and osteoclast related pathological processes, including rheumatoid arthritis, osteo-arthritis, progression of atherosclerotic plaques, and bone metastasis (Ohno H. et al., Mol. Cancer Ther. (2006) 5(11): 2634-2643). c-Kit, the receptor of stem cell factor (SCF), plays a key role in modulation of histamine release from mast cells and influences cell migration and adhesion to the extracellular matrix Inhibition of c-Kit mediates signaling in cynovial tissue from patients with rheumatoid arthritis and induced apoptosis of mast cells. The platelet derived growth factor (PDGF) receptor, which is structurally related to both CSF-1R and KIT, is important for the proliferation and migration of mesenchymal cells and is thought to play a role in the airway remodeling in asthma patients, inflammation in arthritis, and psoriasis. Additionally, Raf-1(c-Raf) inhibition has been shown to suppress smoke-induced airway hyperresponsiveness in mice (Lie Y. et al., Respiratory. Res. (2008) 9(71): 1-10) and has been associated with clinical remission is severe Crohn's disease (Lowenberg M. et al., J. Immunol. (2005) 175:2293-2300).
Additionally, inhibition of the neurotrophin/Trk pathway using NGF antibodies or non-selective small molecule inhibitors of Trk A, B and C has been reported to be effective in treatment of pre-clinical models of inflammatory diseases such as asthma, interstitial cystitis, inflammatory bowel disease, atopic dermatitis and psoriasis (Freund-Michel V., Pharmacology & Therapeutics (2008), 117(1): 52-76; Hu V., The Journal of Urology (2005), 173(3): 1016-21; Di MoIa F. F., Gut (2000), 46(5), 670-678; Dou Y-C., Archives of Dermatological Research (2006) 298(1):31-37. Raychaudhuri S. P., Investigative Dermatology (2004), 122(3): 812-819). PI3K-γ and PI3K-δ have been strongly implicated as a major player in inflammatory conditions (Ruckle T., Nat. Rev. Drug Disc. (2006) 5:903-918; Hawkins P. T., Science (2007) 318:64-66; Barberis L., Thromb Haemost (2008) 99: 279-285.) and tumor growth in a model of colitis-associated cancer (Gonzalez-Garcia A., Gastroenterology (2010) 138:1374-1383). The links between inflammation and proliferative diseases also points to the potential of anti-inflammatory agents as an adjunct to cancer therapy (Karin M., Proc. Am. Thor. Soc. (2005) 2: 368-390; Rakoff-Nahoum s., J. Biol. Med. (2006), 79:123-130; Gust T. C., Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, (2007), 6:19-27; Schmid M. C., Cancer Cell (2011) 19, 715-727). PI3Kγ-deficient mice phenotypes suggest a wide variety of potential therapeutic applications for a selective inhibitor, including: allergic hyper-responsiveness, anaphylaxis, thrombosis, rheumatoid arthritis, glomerulonephritis, systemic lupus erythematosus (SLE), lung injury and airway inflammation related conditions such as COPD, pancreatitis, reduced contractility due to heart failure and ischaemia, and hypertension. Due to system redundancies and the lack of involvement of PI3Kγ in metabolism and house keeping functions, effects induced by PI3Kγ inhibition, which may involve inhibition of chemotaxis and cell specific functions, are seen as soft methods of intervention where undesirable side effects may be minimized (Ruckle T., Nat. Rev. Drug Disc. (2006) 5:903-918).
Neurodegenrative Diseases:
Several kinases are believed to play a role in the pathogenesis of many neurodegenerative disorders. For example, the active form of c-Raf (also termed Raf-1) is upregulated in the brains of Alzheimer's patients and in transgenic Alzheimer's mouse models. The persistent activation of cRaf-1 can activate NFκB and consequently, upregulate the expression of several of its downstream factors such as the amyloid precursor protein (APP), Cox-2 and iNOS. These factors have been found upregulated in numerous neurodegenerative conditions including Alzheimer's, epilepsy, brain trauma, and psychological stress (Burgess S., CNS Neurol Disord Drug Targets. (2010) 1:120-7). In addition, the neurotrophin/Trk pathway, particularly through BDNF/TrkB signaling, has been linked to the etiology of neurodegenerative diseases including Parkinson's disease, multiple sclerosis, and Alzheimer's Disease (AD) (Sohrabji F., Frontiers in Neuroendocrinology (2006), 27(4), 404-414). Several kinases thought to be involved in the underlying inflammatory cause of AD, including GSK3, DAPK1, MAP-kinase, MLCK, and ROCK-1, have been studied. (Villar-Cheda B., Neurobiol Dis. 2012 47(2):268-79) and recently inhibitors of cytokin production in the brain have shown success in models of this disease (Bachstetter A. D., J. Neuroscience, 25 Jul. 2012, 32(30): 10201-10210). Parkinson's Disease (PD) has links to some of the se kinases but the over expression and mutations in LRRK2 has stimulated efforts to inhibit this kinase as a primary target (Kramer T, ACS Chem. Neurosci., (2012), 3(3), 151-160).
Infectious Disease:
The TrkA receptor kinase has been reported to be critical to the disease process in the parasitic infection of Trypanosoma cruzi (Chagas disease) in human hosts (de Melo-Jorge M., Cell Host & Microbe (2007) 1(4):251-261).). Furthermore, a recent study demonstrated in mice that administration of the broad-spectrum receptor tyrosine kinase inhibitor sunitinib blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis (Dalton J. E., J Clin Invest. (2010) 120(4):1204-1216). In addition, sunitinib treatment restored the integrity of the splenic microarchitecture. While this treatment alone was insufficient to cause a reduction in tissue parasite burden, sunitinib proved to be successful as an adjunct therapy by providing dose-sparing effects when combined with an immune-dependent anti-leishmanial drug. These data suggest, therefore, that multi-targeted tyrosine kinase inhibitors may prove clinically useful as agents in the treatment of parasitic infections and perhaps other infectious diseases.
Diabetes:
Imatinib and other TKIs counteract diabetes not only in non-obese diabetic mice, but also in streptozotocin diabetic mice, db/db mice, high-fat treated rats and humans with Type-2 diabetes (T2D). In the later stages of T2D, β-cells are damaged. The molecular events leading to cytokine-induced β-cell dysfunction and death have been linked to the activation of the transcription factors NF-κB (nuclear factor κB) and STATs (signal transducers and activators of transcription). The MAPKs (mitogen-activated protein kinases), such as JNK (c-Jun N-terminal kinase) and p38 MAPK, in response to both cytokines and oxidative stress, play a central role in this chain of events (Eizirik M., Diabetologia (2001) 44(12):2115-2133). Although the mechanisms of protection need to be investigated further, the effects of imatinib and other TKIs in human T2D and the rapidly growing findings from animal models of Type 1 diabetes (T1D) and T2D are encouraging and give hope to improved treatment of this disease (Dariush, 2010). In addition to NF-κB and p38 pathway involvement, PI3K pathways have been implicated as a signaling pathway involved in LPS induced TNF-alpha production in human adipocytes (Hoareau L., Journal of Inflammation (2010) 7:1-12).
Cardiovascular Disease:
Atherosclerosis has also been linked to DDR1 and DDR2 expression. Neointimal thickening is a major cause of restenosis and atherosclerosis and smooth muscle cells (SMCs) are the predominant cell type contributing to its formation after arterial injury. Collagen synthesis by the SMCs after arterial injury acts as an important regulator of the arterial repair through DDR1 and DDR2 activation. In non-human primate hypercholesterolemic diet studies, both DDRs were found to be highly expressed by smooth muscle cells (SMCs) in the fibrous cap of atherosclerotic plaques (Ferri N, Am J Pathol, 2004, 164:1575-1585). Shyu et al demonstrated in balloon injury rat carotid artery model that DDR2 directly promoted the migration and proliferation of vascular smooth muscle cells which contributed to the development of neointimal formation in restenosis and accelerated the arteriopathy. Their study demonstrated that siRNA-mediated inhibition of DDR2 protein expression at the time of balloon injury reduced the neointimal lesion area significantly (Shyu K G, Arterioscler Thromb Vase Biol. 2008; 28:1447-1453). Furthermore, DDR1 null mice had much less severe collagen accumulation in atherosclerotic plaques than wild type mice which was attributed to their considerably lower expression of MMP2 and decreased SMC proliferation and migration (Hou G, J Clin Invest. 2001, 107(6):727; Hou G, Circ. Res 2002, 90:1147-1149).
Several growth factors and growth factor RTKs have been implicated in the abnormal proliferation and migration of pulmonary arterial smooth muscle cells, including PDGF, EGF, FGF, and VEGF (Pullamsetti S. S., PVRIReview (2009), 1(2): 124-128; Hassoun P. M., JACC Vol. 54, No. 1, Suppl S, (2009) S10-19.). Neointimal hyperplasia contributes to atherosclerosis, restenosis after percutaneous coronary intervention, and venous bypass graft disease. Vascular injury in each of these conditions results in the release of mitogenic growth factors and hormones, which contribute to pathological vascular growth. Many of these molecules contribute to neointimal hyperplasia by activating PI3-kinase in vascular smooth muscle cells (Sanada F., Circ. Res. (2009), 105; 667-675) and selective inhibition of PI3K-δ and -γ confers interesting anti-inflammatory effects (Williams O., Chem. & Biol. (2010), 17:123-134). The protein serine/threonine kinases related to ERK-1 and -2 transduce signals to the nucleus not in response to growth factors and other mitogens but in response to cellular stresses such as inflammatory cytokines (IL-1β and TNFα). Ischemia kinases (JNKs) and p38 likely play critical roles in the genetic response of many components of the cardiovascular system disease processes (Force T., Circulation Research. (1996) 78:947-953) and have been suggested as targets for cardiovascular disease therapies (Force T., Circulation, (2004) 109(10): 1196). The Rho-ROCK pathway also has an important role in mediating various cardiac cell functions, including contraction, actin cytoskeleton organization, cell adhesion and motility, proliferation, cytokinesis and gene expression, all of which are involved in the pathogenesis of cardiovascular disease. Abnormal activation of this pathway is associated with the pathogenesis of various cardiovascular diseases such as hypertension, coronary and cerebral vasospasm, restenosis, atherosclerosis, stroke and heart failure, although the roles of the ROCK isoforms (ROCK1 and ROCK2) remain to be elucidated (Shimokawa H., Trends in Pharmacological Sciences, (2007), 28(6):296-302).
Rheumatoid Arthritis (RA):
RA is characterized by leukocyte infiltration, synoviocyte hyperplasia and osteoclastogenesis. Tyrosine kinases have key roles in the signaling pathways that regulate these processes (D'Aura Swanson C., Nat. Rev. Rheumatol. (2009) 5:317-324). Inhibition of receptor tyrosine kinases (RTK) such as platelet-derived growth factor receptors (PDGFR), vascular endothelial growth factor receptors (VEGFR) and Tie receptors have been shown to reduce synovial hyperplasia and angiogenesis (Irvine K. M., FASEB 20 (2006) E-1 to E-12). Non-RTKs are also important in RA. For example, signaling through Burton's tyrosine kinase results in B-cell and T-cell activation while more-specific inhibitors of Janus kinases and Syk, have already shown efficacy in the treatment of RA. Src inhibition is expected to reduce monocyte maturation and osteoclastogenesis. In addition, blocking Kit activation may induce mast cell apoptosis, thereby reducing the production of inflammatory cytokines and degradative molecules in the synovium. The status of current approaches to kinase inhibitor based therapy for RA has been reviewed recently (Muller S., Exprt. Opin. Drug Disco. (2010), 5(9):867-881).
Discoidin Domain Receptors 1 and 2 (DDR1 and DDR2) are collagen receptors with protein tyrosine kinase activity that control fundamental cell processes including cell proliferation, adhesion, migration, and extracellular matrix remodeling (Vogel W, Cellular Signalling, 2006, 18:1108-1116). These RTKs are important in embryonic development, skeletal growth, tissue repair and injury-induced remodeling of blood vessels and the liver (Olaso E, J Clin Invest. 2001, 108:1369-1378; Zhang X H, Arch Med Res. 2010, 41(8):586-92; Ali B R, Hum Mol Gen, 2010, 19(11):2239-2250; Hou G, Circ. Res 2002, 90:1147-1149; Ferri, N, Am J Pathol, 2004, 164:1575-1585). Aberrant activity of both receptors has been linked to human diseases such as lung, kidney and liver fibrosis, atherosclerosis, osteoarthritis, and rheumatoid arthritis. DDR1 and DDR2 have also been implicated in primary and metastatic cancer progression through regulation of metalloproteinase production, cell growth, and chemotactic invasion of normal tissue (Badiola I, Oncol Rep, 2011, 26:971-978).
Hepatic fibrosis in response to chronic injury is similar in all forms of liver disease and involves type I collagen accumulation in the subendothelial spaces between hepatocytes and endothelial cells. The newly generated fibrillar collagen replaces basement membrane like matrix containing type IV collagen. This conversion to fibrillar collagen is pivotal in mediating the loss of differentiated function that characterizes progressive liver disease. Liver stellate cells are the major source of fibrosis as they convert from quiescent cells to proliferative and fibrogenic myofibroblasts. In liver fibrosis mouse models, DDR2 is upregulated in stellate cells following increased collagen synthesis and is an inducer of MMP-2 mediated growth stimulation suggesting this collagen receptor may help perpetuate the fibrosis (Olaso E, J Clin Invest. 2001, 108:1369-1378). DDR2 has also been found at increased levels in the mesenchymal compartment as well as the biliary epithelial cells in cirrhotic livers (Mao T K, Autoimmunity 2002, 35(8):521.).
DDRs have also been shown to play a role in fibrosis of the kidney and lung. DDR1-null mice have also been found to have significantly reduced fibrotic and inflammatory responses in kidney hypertension models (Vogel W, Cellular Signalling, 2006, 18:1108-1116.). The DDR1b isoform was found to be selectively induced in idiopathic pulmonary fibrosis (IPF) patients during disease progression and high levels of DDR1 can be found in CD14 positive cells from bronchioalveolar lavage fluid from these patients compared to healthy volunteers or patients with other lung diseases (Matsuyama W, FASEB J, 2003, 17(10):1286).
The discoidin domain receptors are also associated with inflammation and arthritis. LPS and IL-1β induces monocyte and neutrophil expression of the DDR1a and DDR1b isoforms. Transfection of DDR1a into leukemia cell lines promotes adhesion while DDR1b enhances monocyte differentiation to macrophages and upregulates their MIP-1α and MCP-1 production during extravasation (Matsuyama W, J Immunol. 2005 174(10):6490). DDR1 is upregulated in activated T cells and can act as a co-stimulator under suboptimal TCR/CD3 activating conditions (Dang N, J Immunother. 2009, 32(8):773-784). The receptor kinase also enhances primary human T cell migration through 3D collagen by a mechanism not dependent on adhesion (Hachehouche L N, Mol Immunol. 2010, 47(9):1866-1869; Chetoui N, J Cell Biochem. 2011, 112(12)3666-3674).
DDR2 has been found to be integral in the maintenance and progression of osteoarthritis and rheumatoid arthritis. DDR2 mediated MMP-13 induction exacerbates the articular cartilage degeneration found in osteoarthritis patients. Reports both in mouse arthritis models and from human knee joints found a correlation between increased DDR2 and MMP-13 expression and the degree of type II collagen breakdown. These results suggest the perpetuation of DDR2 activation becomes a vicious circle where by DDR2 promotes tissue catabolism which leads to cartilage damage and further DDR2 upregulation and activation (Sunk I G, Arthritis & Rheumatism, 2007, 56(11):3685-3692.). Xu and his colleagues demonstrated that reducing DDR2 expression by using DDR2−/+ heterozygous mutant mice led to decreased articular cartilage degeneration of the knee joints induced by injury or type XI collagen deficiency (Xu L, Arthritis & Rheumatism 2010, 62(9):2736-2744). These data suggest that regardless of the initiating event, osteoarthritis disease progression is perpetuated by the continued activation of DDR2 and therefore therapeutic agents that specifically inhibit this kinase may be successful agents in the prevention and treatment of osteoarthritis.
The preponderance of evidence from research on discoidin domain receptor function demonstrates that DDRs are molecular sensors that monitor extracellular matrix integrity. However, aberrant or uncontrolled DDR1 and DDR2 signaling has been associated with a variety of illnesses such as arthritis, fibrotic disorders and cancer highlighting the potential importance of these collagen receptors in human health and disease. These data suggest DDR1 and DDR2 may be good targets for therapeutic intervention in multiple indications.
Pain:
Tropomyosin-related Kinases (Trk's) are the high affinity receptor tyrosine kinases activated by a group of soluble growth factors called neurotrophins (NT). There are 3 Trk receptor family members: TrkA, TrkB and TrkC. Trk's are widely expressed in neuronal tissue and are important in the maintenance, signaling and survival of neuronal cells (Patapoutian A., Current Opinion in Neurobiology, (2001), 11, 272-280). Inhibitors of the Trk/neurotrophin pathway have been shown to be effective in many pre-clinical animal models of pain. For example, antagonistic NGF and TrkA antibodies have been shown to be efficacious in inflammatory and neuropathic pain models (Woolf C. J., Neuroscience (1994), 62:327-331. Zahn P. K., J. Pain (2004), 5:157-163; Shelton D. L., Pain (2005), 116:8-16; Delafoy L., Pain (2003) 105:489-497; Theodosiou M, Pain (1999) 81:245-255; Li L., Mol. Cell. Neurosci. (2003), 23, 232-250; Gwak Y. S., Neurosci. Lett. (2003), 336: 117-120). Furthermore, several groups have demonstrated that BDNF levels and TrkB signaling is increased in the dorsal root ganglion after inflammation (Cho H. J., Brain Res (1997) 764: 269-272.) and antibodies that decrease signaling through the BDNF/TrkB pathway inhibit neuronal hypersensitization and the associated pain (Li C-Q., Molecular Pain, (2008), 4(28), 1-11).
It has also been reported that NGF secreted by tumor cells and tumor invading macrophages directly stimulates TrkA located on peripheral pain fibers. Using various tumor models in both mice and rats, it was shown that neutralizing NGF antibodies inhibit cancer related pain to a degree equal to or better than the highest tolerated dose of morphine. In addition, activation of the BDNF/TrkB pathway has been implicated as a modulator of neuropathic, inflammatory and surgical pain (Matayoshi, J. Physiol. (2005), 569:685-95; Thompson S. W. N., Proc. Natl. Acad. Sci. USA (1999), 96:7714-18; Li C-Q., Molecular Pain, (2008), 4(28), 1-11). These bodies of data suggest inhibitors of TrkA and/or other Trk kinases may provide an effective treatment for chronic pain states.
Kinases in Cancer:
Although kinase mediated pro-inflammatory or wound healing signaling pathways play important support roles in cancers (Karin M., Proc. Am. Thor. Soc. (2005) 2: 368-390; Rakoff-Nahoum S., Yale J. Biol. Med. (2006), 79:123-130; Gust T. C., Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, (2007), 6:19-27), these processes are usually not sufficient to initiate tumorigenesis. More often, the transformation process requires the aberrant activation of or activating mutation in kinases involved in other tumor specific key signaling pathways. The term ‘oncogenic addiction’ is often used when gene mutations provide a survival advantage for tumor cells over non-transformed cells and the expression of that gene product is required to avoid cell death. These oncogenes are commonly receptor tyrosine kinases (e.g. EGFR, PDGFRA, MET) or kinases in the PTEN/PI3K/AKT or Ras/Raf/MEK/ERK signaling pathways. For example, it has been estimated that 88% of all glioblastomas have altered signaling in one of these kinase pathways (Cancer Genome Atlas Research Network, Nature; (2008), 455:1061-1068).
The Ras/Raf/MEK/ERK and PI3K/Akt Pathways:
The RAS/RAF/MEK/ERK and the RAS/PI3K/PTEN/mTOR kinase cascades are two key pathways that contribute to many cancers that are illustrative of the compensatory crosstalk and redundancies in signalling networks that can lead to development of innate or acquired resistance to their individually targeted therapies, FIG. 1 (Gibbony G. T. and Smalley K. S. M., Cancer Discovery (2013) 4(3): 260-263).
The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to different growth signals and is frequently dysregulated in cancer. The RAF family proteins are serine/threonine specific kinases and are key players in the MAPK pathway. These proteins act immediately downstream of Ras to conduct extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events. Thereby cell growth, proliferation, and differentiation can be regulated in response to growth factors, cytokines, and hormones (Christensen C., Oncogene (2005), 24(41):6292-6302. Schnidar H., Cancer Res. (2009), 69(4):1284-1292.). The Ras/Raf/MEK/ERK pathway has been found to be upregulated in approximately 30% of all cancers with higher percentages seen in cutaneous melanomas as well as colon, lung, ovarian, and kidney tumors (Hoshino R. Oncogene, (1999) 18:813-822). Mutated RAS, especially KRAS, is seen in over 20% of all human cancers (Bamford S., Br. J. Cancer 91 (2): 355-358; Bos J. L., A Review Cancer Res (1989) 49(17):4682-4689). RAS mutations have also been shown to lead to the promotion of PI3K signaling and dysregulation of the downstream RAF/MEK/ERK signaling pathways. Although early attempts to target Ras have not yielded any viable drug candidates, many novel compounds inhibiting the activities of Raf and MEK have been developed and investigated in clinical trials in recent years. Although the first MEK inhibitor (CI-1040) lacked efficacy in clinical trials, its low toxicity has encouraged the search for novel compounds with enhanced target potency (Wong K-K, Recent Patents on Anti-Cancer Drug Discovery, (2009), 4:28-35).
The three Raf kinases are designated as A-Raf, B-Raf, and C-Raf. At this time only B-Raf (v-Raf murine sarcoma viral oncogene homologue B 1) is frequently found mutated in various cancers (Palanisamy N., Nature Medicine (2010), 16(7):793-798.). The most common B-Raf mutation constitutes 90% of all mutations to this kinase. The substitution of a glutamic acid residue for a valine moiety at codon 600 (V600E) results in a constitutively activated kinase that is ˜500-fold more active than the wild-type protein (Hoeflich K. P., Methods in Enzymology (2008), 439: 25-38). This mutation, which occurs with a frequency of 50-70% in cutaneous malignant melanoma, is also present in a wide range of other human cancers, particularly thyroid (30%), colorectal (10%), and ovarian (35%) cancers (Flaherty K T, et al., NEJM (2010) 363:809-819; El-Osta H, et al., PLoS ONE (2011) 6(10):e258060). Advanced malignant melanoma has a tendency to rapidly metastasize throughout the body and develop resistance to treatment. In addition, melanoma rates continue to rise and the average patient age continues to decrease. Observation that inhibition of B-Raf signaling blocks cancer cell proliferation and induces apoptosis and its dysregulation in multiple tumor types validates V600E B-Raf as an important therapeutic target with excellent opportunities for anticancer drug development. Increased phosphatidylinositol 3-kinase (PI3K) signaling is also prevalent in many types of cancer (Vivanco I., Nat Rev Cancer (2002), 2(7):489-501; Serra V., Oncogene (2011) 2; 30(22):2547-57). Dysregulation of this pathway may be caused at a molecular level by activating mutations of PI3K itself, by loss of PTEN, a negative regulator of PI3K activity, mutations in regulatory proteins, or by a variety of factors both up and downstream of PI3K. PTEN is one of the most commonly mutated or deleted genes in human cancer, second only to p53 (Cantley L. C., Proc Natl Acad Sci USA. (1999) 96(8):4240-4245), and somatic mutations of the PI3K p110α chain are found in 30% of all epithelial cancers (Engelman J. A., Clin Cancer Res 2008; 14:2895-2899). In addition, P70S6K1, a kinase downstream of PI3K/AKT pathway that is principle to the expression of VEGF and survivin, has become a target of recent interest for cancer therapy (Skinner H. D., J. Boil. Chem. (2004) 279(44): 45643-45651; Zhao P., Biochem. Biophys. Res. Commun. (2010), 395(2): 219-224). Finally, activation of the PI3K/AKT pathway has been strongly implicated in escape mechanism that compromise the effectiveness of specific kinase targeted therapies (Wee S., Cancer Res. (2009) 69(10) 4286-4293; Hynes N. E, Cancer Cell (2009)15: 353-355; Villanueva J., Cancer Cell (2010) 15(6)):683-695; Paraiso K. H. T., Cancer Res. (2011) 71(7): 2750-60).
Stimulation of the Ras/PI3K/PTEN/AKT/mTOR pathway and hyper-activation of the Ras/Raf/Mek/Erk axis are dominant compensatory mechanism by which inhibition of B-Raf(V600E) is ultimately circumvented (Davies M A et al., Cancer J. (2012) 18(2):142-7, Steelman L S, et al. J Cell Physiol. (2011) 226(11):2762-81; Yajima J, et al., Dermatology Research and Practice 2012; Article ID 354191), FIG. 1. Mutations that result in loss of PTEN function, activation of Ras, and/or loss of the RAS suppressing effects of neurofibromin (via the NF1 gene) have been identified as major contributors to both the innate and acquired resistance to current front line B-Raf inhibitor therapies, FIG. 1 (Maertens O., Cancer. Discovery. (2013) 3(3); 338-49; Gibbony G T and Smalley K S M: 2013.
Other Important Receptor Kinases in Cancer:
Kinases upstream or outside of the Ras/Raf/Erk and PI3K/Akt pathway have also been implicated in cancer cell differentiation and proliferation including the receptor tyrosine kinases in the Axl/Mer/TYRO3 and the Trk neurotrophin receptors (TrkA, TrkB TrkC) families.
The Axl/Mer/Tyro3 kinase family members have been implicated in tumor cell proliferation, cell-cell interactions, and cell migration and invasion, suggesting multiple roles for this pathway in tumorigenesis.
Axl and Mer are expressed in various organs including the brain and testes during development (Nagata K, J. Biol. Chem. (1996) 271 (47): 30022-30027.). However in human adults their expression, which is normally very low, returns to high levels in a variety of tumors including glioblastoma, pancreatic, lung, thyroid, hepatocellular, colon, renal, gastric, and breast carcinomas (Funakoshi H., J. Neurosci. Res. (2002) 68:150-160; Li Y., Oncogene (2009), 28:3442-3455. Challier C., Leukemia (1996) 10:781-787; Craven R. J., Int. J. Cancer (1995) 60:791-797; Vajkoczy P., Proc. Nat. Acad. Sci. (2006):103(15): 5799-5804; Sheih Y-S., Neoplasia. (2005) 7(12): 1058-1064; Xianzhou S., Cancer (2011), 117(4):734-743). Tyro3 is also expressed in the brain and testes and has also been linked to NK cell differentiation. Recently Tyro3 has been identified as the upstream regulator of microphthalmia-associated transcription factor (MITF), the ‘lineage addiction’ oncogene in malignant melanoma. In animal models, blocking Tyro3 repressed cellular proliferation and colony formation in melanoma cells thereby inhibiting tumorigenesis in vivo (Zhu S., Proc. Nat. Acad. Sci. (2009) 106(4):17025-17030). Axl, Mer, and Tyro-3 mediate multiple oncogenic phenotypes and activation of these receptor tyrosine kinases has been shown to provide a mechanism of chemoresistance in a variety of solid tumors. The role of Axl and Gas6 in downstream signaling leading to drug resistance involves a cancer cell's transition from an epithelial phenotype to one with mesenchymal properties (epithelial-to-mesenchymal transition or EMT). The EMT process allows a cancer cell to acquire many of the hallmarks required for oncogenesis and drives the cell into a state that is more resistant to therapy. The literature suggests that selective inhibition of Axl signaling reverses EMT (Byer L A, et. al., Clin Cancer Res. (2013) 19(1):279-290) and shifts the cell back into a sensitive state which can then respond to targeted therapy. Targeted inhibition of these RTKs may be effective as anti-tumor and/or anti-metastatic therapy, particularly if combined with standard cytotoxic therapies (Linger R. M. A., Targets (2010) 14(10):1073-1090).
The Trk family of neurotrophin receptors are crucial for the normal development of the peripheral nervous system. These receptor tyrosine kinases signal through the PI3K, Ras/Raf/MEK and PLCγ1/PKC pathways and have been found to play a critical role in neuroblastomas, the most common and deadly solid tumor in children (Brodeur G. M., Clin Cancer Res (2009) 15(10): 3244-3250). The Trk isoform expressed by the neruoblastoma can be prognostic as TrkA and TrkC expressing tumors are more prone to spontaneous regression and a more favorable outcome whereas TrkB are more often very aggressive and frequently have concomitant MYCN amplification. TrkB has been shown to suppress anoikis, or cell death induced by cell detachment, and thereby allowing the metastatic spread of tumor cells (Geiger T. R., Cancer Res (2007) 67(13):6221-9). Trk family gene rearrangements or aberrant expression have also been identified in papillary thyroid carcinomas, breast cancers, non-small cell lung cancer, prostate cancer, pancreatic ductal adenocarcinoma, pediatric sarcomas, and leukemias (Tognon C., Cancer Cell (2002), 2:367-76; Liu Q., EMBO J (2000); 19: 1827-38; Eguchi M., Blood (1999), 93:1355-63; Harada T., Clin Cancer Res. (2011), 17(9):2638-45. Jones-Bolin S. E., Proc Amer Assoc Cancer Res (2005) 46: Abstract #3026).
Osteolytic metastases are common in many types of cancer and have been found in up to 70% of patients with advanced breast or prostate cancer and in approximately 15% to 30% of patients with lung, colon, stomach, bladder, uterus, rectum, thyroid, or kidney carcinomas. Bone metastases can cause severe pain, hypercalcemia, pathologic fractures, spinal cord compression, and other nerve-compression syndromes. Expression of TrkA and TrkC receptor kinases have been observed in the bone forming area in mouse fracture models and NGF expression was observed in almost all bone forming cells (Asaumi K., Bone (2000) 26(6): 625-633.). These data support exploring the use of pan Trk inhibitors for the treatment of bone remodeling diseases such as bone metastases in cancer patients as well as osteoporosis and rheumatoid arthritis.
Discoidin Domain Receptors 1 and 2 (DDR1 and DDR2) are collagen receptors with protein tyrosine kinase activity that control fundamental cell processes including cell proliferation, adhesion, migration, and extracellular matrix remodeling (Vogel W, Cellular Signalling, (2006) 18:1108-1116). These RTKs are important in embryonic development, skeletal growth, tissue repair and injury-induced remodeling of blood vessels and the liver (Olaso E, J Clin Invest. (2001) 108:1369-1378; Zhang X H, Arch Med Res. 2010, 41(8):586-92; Ali B R, Hum Mol Gen, (2010) 19(11):2239-2250; Hou G, Circ. Res (2002) 90:1147-1149; Ferri, N, Am J Pathol, (2004) 164:1575-1585). Aberrant activity of both receptors has been linked to human diseases such as lung, kidney and liver fibrosis, atherosclerosis, osteoarthritis, and rheumatoid arthritis. DDR1 and DDR2 have also been implicated in primary and metastatic cancer progression through regulation of metalloproteinase production, cell growth, and chemotactic invasion of normal tissue (Badiola I, Oncol Rep, (2011) 26:971-978).
DDR1 and DDR2 have been linked to several human cancers. DDR1 has been found in breast, ovarian, brain, esophageal, lung and immune system cancers (Vogel W, Cellular Signalling, 2006, 18:1108-1116).). Barker et al demonstrated DDR1 was more highly expressed in cancerous breast epithelial cells than in adjacent normal breast tissue (Barker K T, gene, 1995 10:569). This collagen receptor has also been shown to be a direct transcriptional target for the p53 tumor suppressor gene. DDR1 inhibition in tumor cells with wild type p53 activity results in increased apoptosis (Ongusaha P P, EMBO J, (2003) 22(6): 1289). DDR2 has been shown to play a role in breast, lung and immune system cancers as well. Recently a group reported that approximately 3% of 277 lung squamous cell carcinoma patients had DDR2 mutations (Kotz J, SciBX 2011, 4(20):1-2). Though DDR1 and DDR2 have not been shown to be oncogenes in carcinogenesis, they likely act through regulating tumor cell growth, adhesion and metastasis by controlling collagenous extracellular matrix remodeling and metalloproteinase expression.
Other Non-Receptor Tyrosine Kinases in Cancer:
Several non-receptor tyrosine kinases such thirty-eight-negative kinase 1 (Tnk1), JAK kinases, breast tumor kinase (Brk or PTK6), ROS, and ARG have also been implicated in tumor progression, survival and metastasis. Though the tyrosine kinase Tnk1 has been identified as a tumor suppressor gene in some cellular contexts, recently a novel gene translocation has been identified that results in a fusion protein combining part of C17ORF61 with Tnk1 kinase (Gu T-L., Leukemia (2010), 24:861-865.). The TNK1-C17ORF61 fusion protein, which retains constitutive Tnk1 tyrosine kinase activity, was confirmed to drive the proliferation and survival of Hodgkin's lymphoma (HL) cell line, L-540. In addition, the application of functional genomics by using HT-RNAi screens has allowed researchers to identify TNK1 as a growth-associated kinase in pancreatic cancer cells (Henderson M. C., Mol Cancer Res. (2011) 9(6).).
Activating mutations in JAK family members are observed in leukemias and myeoloproliferative neoplasms (Verstovsek S., Hematology (2009) 636-642). Several lines of evidence support the conclusion that JAK/STAT signaling is exaggerated in hematological malignancies and likely contributes to disease pathogenesis. Activating mutations in Jak1 have been described in acute lymphoblastic liekemia (ALL) and the Jak2V617F mutation is particularly important in myeleoproliferative neoplasms (MPNs) and myelofibrosis.
Brk is a member of a novel family of soluble protein tyrosine kinases, considered to be distantly related to c-Src (Ostrander J. H., Cancer Res (2007); 67: 4199-4209). Brk has been shown to localize to the nucleus of some breast and prostate cancer cell lines and is coamplified and coexpressed with ErbB2 in human breast cancers. Brk has been shown to interact with EGFR and ErbB3 and the expression of Brk enhances EGF-induced ErbB3 phosphorylation and the recruitment of p85 phosphatidylinositol 3-kinase to ErbB3, which potentiates PI3K activity (Xiang B., Proc. Nat. Acad. Sci. (2008); 105(34): 12463-12468). Data from these recent studies place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation.
ROS kinase is one of the last remaining orphan receptor tyrosine kinases with an as yet unidentified ligand and the normal function so this kinase in different body tissues have not been fully identifies. However, ectopic expression, as well as the production of variable mutant forms has been reported in a number of cancers, such as glioblastoma mutifore and non-small cell lung cancer, suggesting a role for ROS kinase in deriving such tumors. The recent discovery of new selective inhibitors for ROS, along with the development of new diagnostic tools for the detection of ROS fusion proteins, indicates that targeting of this kinase and its mutant forms may have clinical applications for the treatment of cancers (El-Deeb I. M. et al., Medicinal Research Reviews, (2011) 31(5) 794-818).
ARG is an ABL-related kinase very similar to c-ABL at the SH3, SH2, and kinase domains and is expresses widely in normal cells (Krushe G. D., Science (1986) 234:1545-1548; Perego R., Oncogene (1991) 6, 1899-1902). ARG is also implicated in leukemogenesis by the fusion between ARG and ETV6 (ETS translocation variant 6), also known as TEL (translocation ETS leukemia). The ARG:ETV6 fusion was identified in two independent cases of human leukemias with t(1;12)(q25;p13) translocation (Cazzaniga G., Blood, (1999). 94:4370-4373; Iijima Y. Blood, (2000) 95: 2126-2132) and in a T-lymphoblastic cell line derived from a patient with acute lymphoid leukemia carrying t(1;10;12)(q25;q23;p13) (Nishimura N., Oncogene (2003) 22: 4074-4082). Constitutive activation of ARG and Abl kinases has also been implicated in the promotion of breast cancer cell invasion (Srinivasan D., Cancer Res (2006) 66(11): 5648-55). It has been reported that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571 (Okuda K., Blood (2001), 97:2440-2448) which may contribute to the ability of STI571 (Imatinib/Gleevec) to induce hematologic remission in most patients with chronic myeloid leukemia.
Roles of Escape Mechanisms in the Treatment of Cancer:
Targeted inhibitors against specific tyrosine kinases known to be critical in tumor cell growth, differentiation, and survival have generated a lot of excitement over the last decade. Although there have been some dramatic examples of clinical responses in tumors known to have genetic mutations in single genes, i.e. the BCR:Abl fusion protein in CML and the B-Raf (V600E) mutation, highly specific kinase inhibitors can be met ultimately with resistance and tumor escape due to pathway enabling mutations in the target or suppressor proteins and up-regulation of compensatory proteins or pathways. In spite of recent advances, improving the outcomes for patients afflicted with relapsed and refractory cancer still represents a significant challenge. Too often, newly approved, targeted agents produce a significant upfront response in cancer patients only to be followed by drug resistance and progressive disease. Significant efforts have been made to understand the mechanisms of drug resistance, particularly to targeted agents.
Tumor cells that harbor B-Raf(V600E) exhibit oncogenic addiction and targeted inhibitors, such as the Type-I inhibitor Vemurafenib, have demonstrated remarkable efficacy in advanced stage disease driven by this mutation (Ribas A, et al., Clin Oncol (2011) 29:Suppl:8509; Chapman P. B., et al. New Engl. J. Med. (2011) 364: 2507-2516]. However, resistance to Raf inhibitors, such as Vemurafenib, develops quickly (within 6-7 months) and recent studies have suggested that drug addiction pays a role and that removal of drug may be required to halt this life threatening resistance (Das M. et al. 2013 e-print, doi:10.1038/nature11814). In addition, cell population heterogeneity, compensatory pathway activation, inactivation of suppressor proteins, and external stimulation by the micro-environment can conspire to promote resistant disease, FIG. 1 (Gibbony G. T. and Smalley K. S. M., Cancer Discovery (2013) 4(3): 260-263; Paraiso K H T, et al., Clinical Cancer Research, (2012) 18(9):2502-2514.).
Another limitation of Type-I, ATP competitive, B-Raf inhibitors have is due to their ability to transactivate wild-type B-Raf and Raf-1 in normal cells (Hatzivassiliou G, et al Nature, (2010) 464:431-435; Heidorn S J, et al. Cell (2010) 140:209-221; Poulikakos P I, et al. Nature 2010 464:427-430) and intermittent treatment has again been proposed as a means of improving patient outcomes (Thakur M D, et al., Nature (2013) 494: 251-255). Therefore, efforts continue to understand the limitations of current targeted therapies and escape mechanisms with improved inhibitors and adjunct therapies.
Recently, new insights into the mechanisms of resistance have been provided (Maertens O. et al., Cancer Discovery (2012) 3(3): 338-349; Whittaker S. R. et al., Cancer Discovery (2012) 3(3): 350-362; Gibney G. T. and Smalley K. S. M., Cancer Discovery (2013) 4(3): 260-263). Although B-Raf mutations play a well established role in melanogenesis, without additional genetic alteration, tumor development is often restricted to oncogene-induced senescence (OIS). Nf1 mutations suppress B-Raf induced senescence, promote melanocyte hyperproliferation, and enhance melanoma development. Nf1 mutations function by deregulating both PI3K and ERK pathways. As such, Nf1/B-Raf mutant tumors are resistant to B-Raf inhibitors but are sensitive to combined inhibition of MAPK/ERK and mTOR. If Nf1 is mutated or suppressed in human melanomas that harbor concurrent B-Raf mutations, the Nf1 ablation decreases the sensitivity of melanoma cell lines to B-Raf inhibitors. Importantly, loss of Nf1 activity is seen in patients following sustained treatment with B-Raf inhibitors and mechanisms of Nf1 inactivation have been associated with acquired or innate resistance to these targeted therapies in melanoma.
Constitutive activation of signaling upstream or further downstream from the inhibited target protein is a common resistance mechanism. For example, blockade of mTOR with rapamycin analogs results in an increase in AKT signaling that reduces their overall therapeutic effect (Zitzmann K., Cancer Letters (2010), 295(1): 100-109; O'Reilly K. E., Cancer Res. (2006), 66: 1500-1508). In such cases, targeting multiple kinases in the affected signaling pathway can maximize pathway inhibition. Consequently, mixed inhibitors of PI3K and mTOR have been developed (Brachmann S., Curr. Opin. Cell Biol. (2009) 21(2): 194-198; Venkatesan A. M., Bioorg. Med. Chem. Lett. (2010), 20(2): 653-656).
Another common cause of specific inhibitor resistance is through the activation of a redundant receptor or parallel pathway that can functionally substitute for the inhibited one. This type of resistance occurs with receptor tyrosine kinases when related family members can perform overlapping functions and inhibiting one receptor cannot completely block downstream signaling. It has been found that approximately 20% of tumor samples from patients that became resistant to EGFR inhibitors had MET gene amplification (Engleman J. A., Clin Cancer Res 2008; 14:2895-2899). The crosstalk between EGFR and MET, observed in breast cancer cells, explains the EGFR inhibitor resistance seen in such tumors (Tao Y., Nat. Clin. Pract. Oncol, (2007) 4(10): 591-602). Therefore an inhibitor that inhibits both EGFR and MET could be efficacious in treating such patients. Up-regulation of the PI3K/PTEN signaling through PIK3CA activating mutations or PTEN loss is another mechanism found in EGFR inhibitor resistance (Janmaat J. L., Clin Cancer Res, (2003), 9(6):2316-2326). Chemotherapy resistance has also been tied to PI3K/Akt activation through EGFR (Winograd-Katz S., Oncogene (2006), 25:7381-7390).
Of particular interest is the crosstalk between the PI3K/AKT/mTOR and RAS/Raf/MEK/ERK pathways often utilized by tumors as a compensatory mechanism when specific inhibitors of a single pathway are used (Faber A. C., Cell Cycle (2010) 9(5) 851-852). It has been shown that PI3K inhibition in HER2-overexpressing breast cancers can lead to the up-regulation of the compensatory ERK signaling pathway. Inhibition of both PI3K and MEK simultaneously has been demonstrated to lead to decreased proliferation and superior anti-tumor activity in animal models and this combination therapy is currently being studied in the clinic (Worcester S., Elsevier Global Medical News. (2011) Apr. 11).
As described above, the issues surrounding the mono-specific TKIs has led to the revitalization of interest in development of ‘dirtier’ kinase inhibitors that hit multiple kinases at the same time. One early successful multikinase inhibitor is Sorafanib (Naxavar) which targets Raf, VEGFR, PDGFR(3, FLT3, p38 and c-Kit all with IC50s in the nanomolar range. Other examples of approved multikinase inhibitors include Sunitinib (Sutent), Erlotinib (Tarceva) and Imatinib (Gleevac). However, these early versions of multikinases rarely hit both the Ras/Raf/ERK and PI3K/AKT/mTOR pathways at the same time.
It has been reported that tumors, such as AML acquire resistance to these multikinase Raf/Flt3/c-Kit inhibitors, due to the activation of compensatory PI3K/AKT pathways after several months of treatment. It is becoming increasingly apparent that inhibiting both the target oncogene and kinases involved in the commonly used escape mechanisms will be required to achieve durable responses with targeted cancer therapies. Single target TKIs currently in phase 1 or phase 2 clinical trials are providing significant amounts of data on which pathways are commonly dysregulated in the most prevalent tumor types and more importantly which compensatory pathways lead to tumor escape. This information will help determine which specific inhibitors would be most effective given in combination leading to more durable tumor growth inhibition in the patients.
Another reoccurring mechanism that appears to be an underlying cause to both upfront and acquired resistance to many receptor tyrosine kinase (RTK) inhibitors is the up-regulation of Axl. This has been demonstrated in multiple cancer types with numerous targeted agents, including imatinib resistance in gastrointestinal stromal tumors (GIST), erlotinib resistance in non-small cell lung cancer (NSCLC) (Byers, et. al., Clin Cancer Res. 2013; 19(1):279-290), PKC412 resistance in acute myeloid leukemia (AML), cetuximab resistance in squamous cell carcinoma of the head and neck (SCCHN) (Giles, et al., Mol Cancer Res (2013) 12(11):2541-2558), and lapatinib resistance in breast cancer (Liu L, et al., Cancer Res (2009) 69(17):6871-6878. Holland S, et. al. (Cancer Res. 2010) 70:1544-1554). Axl is also one of the most common RTKs detected in breast cancer (Meric F, et. al., Clin Cancer Res. (2002) 8:361-367) where expression promotes metastasis and is associated with a poor prognosis (Gjerdrum C, et. al., Proc Natl Acad Sci USA. (2010) 107:1124-1129). Moreover, inhibition of Axl has been shown to restore sensitivity to targeted agents in a synergistic manner (Verma A, et. al., Mol Cancer Ther. (2011) 10(10); 1763-73). Axl is a member of the TAM receptor kinase family that includes Mer and Tyro3. The over expression of any of the three family members has been associated with tumor cell survival and growth, increased migration, and angiogenesis (Linger R M, Adv Cancer Res. (2008) 100:35-83 and Linger R M., Expert Opin Ther Targets. (2010) 14:1073-1090). AML cells are known to induce the expression and secretion of the TAM receptor ligand Gas6 (growth arrest-specific gene 6) by bone marrow-derived stroma cells, which in turn mediates proliferation, survival and chemo-resistance in AML cells. Mer receptor tyrosine kinase over expression has been shown to contribute to leukemogenesis (Lee-Sherick A B, Oncogene, (2013) 32(46):5359-68) and its inhibition increases chemo-sensitivity and decreases oncogenic potential in T-cell acute lymphoblastic leukemia (Brandao L N, Blood Cancer Journal, (2013) 3 (1): e101 DOI: 10.1038/bcj.2012.46). A good case has also been made that Axl, Mer and Tyro3 are potential targets in Melanoma and as an adjunct to immunotherapies (Sensi M, et al, J. Invest. Derm. (2011) 131:2343-57; Schlegel J, et. al., J Clin Invest. (2013) 123(5):2257-2267; Demarest S J, et. al., Biochemistry. (2013) 52(18):3102-18).
Deregulation of protein synthesis is also a common event in human cancers. A key regulator of translational control is elF4E and reports indicate that eIF4E activity is a key determinant of both Ras/PI3K/Akt/mTOR and Ras/Raf/Mek/Erk mediated tumorigenic activity. Because activation of eIF4E involves phosphorylation of a key serine (Ser209) specifically by MAP kinase interacting ser/thr kinases (Mnk1 and Mnk2) (Hou J., Oncotarget (2012) 3:118-131), efforts to discover either selective or combined targeted inhibitors of these kinases are underway by several research groups (Kassoum N, et. al.—2013). Indeed, Mnk inhibition by the antifungal agent Cercosporamide suppresses primitive leukemic progenitors (CFU-L) from AML patients in a dose-dependent manner (Altman J K, Blood (2013) 121(18) 3675-3681). Resistance in chronic myeloid leukemia (CML) is also facilitated through elF4E over expression by blast-crisis granulocyte macrophage progenitors (GMPs) which then act as leukemia stem cells (LSCs) (Smith C C, Hematology Am Soc Hematol Educ Program. (2011) 2011:121-7). Although elF4E activation is necessary for oncogenic transformation, it seems dispensable for development of normal hematopoietic stem cells (HSCs).
Moreover, since Mnks act downstream of both MAPK and PI3K pathways, their inhibition may also have utility in Vemurafenib resistant cancers where Raf up-regulation and aberrant Ras/PI3K/Akt/mTOR axis activity conspire to promote resistant disease within 6-7 months (Davies M A, Cancer J. (2012)18(2):142-7). Simultaneous inhibition of both TAM and Mnk family members should be more effective as an adjunct to targeted therapies than either TAM or Mnk inhibition alone and data suggests that such an agent may not significantly increase the side effect burden of targeted therapies (Linger R M., Expert Opin Ther Targets. (2010) 14:1073-1090).
Applications of Imaging Agents:
Use of imaging agents for monitoring disease progression is well established (Smith-Jones K. M., J. Nuclear Medicine (1994), 35(2): 219-325; Solit D. B, Cancer Res (2007), 67(23):11463-11469). Interest has intensified regarding the application of such agents for the diagnosis, localization, and characterization of cancers (Hoffman J. M., Radiology (2007) 244(1): 39-47; Stehouwer J. S., J. Med. Chem. (2010), 53(15): 5549-5557) as well as both acute and chronic inflammatory and degenerative diseases. More recently, applications directed specifically at the monitoring of kinase activity have also been reported (Dumont R. A., Cancer Res. (2009), 69(7): 3173-3179; Samen E., Eur. J. Nucl. Med. Mol. Imaging (2009), 36:1283-1295; Pisaneschi F., Bioorg. Med. Chem. 2010, 18: 6634-6645; Koehler L., European Journal of Medicinal Chemistry (2010) 45: 727-737.). The promise of imaging technologies for improved benefit, reduced cost, and personalization of medicine is significant.
General Construction of Kinase Inhibitors:
The general construction strategies and key structural elements for kinase inhibitors have been analyzed and reviewed extensively (Liu Y., Nature Chemical Biology (2006) 2:358-364; Goshe A. K. J. Med. Chem. (2008), 51(17):5149-5171; Zhang J., Nat. Rev. Cancer (2009) 9(1): 28-39) and, based on their mechanism of inhibition, they can be classified as either of five types, (Cozza G., Anti-Cancer Agents in Medicinal Chemistry, 2009, 9:778-786).
Type-I inhibitors compete at the ATP binding site of a kinase and typically bind to three subsites: 1) the purine binding site or “Hinge Region”, 2) the solvent exposed “Flap Region” at the entrance to the ATP site, and 3) a lipophilic site adjacent to the purine site that is often referred to as the “Gatekeeper Region”. Taken together, these binding sites recognize “Hinge-Gatekeeper Motifs” (HGM) that can achieve useful selectivity and profiles of inhibition, FIG. 2A. One major disadvantage of Type-I, ATP competitive, inhibitors is the kinetic challenge resulting from the millimolar physiological concentrations of ATP. The second challenge is achieving selectivity for a particular kinase since the basic construction, functionality, and topography of ATP binding sites are necessarily similar.
In contrast, Type-II inhibitors bind to an alternate inactive conformation, exhibited by some kinases, in which a conserved Aspartyl-Phenylalanyl-Glycine (DFG) containing loop is reoriented such that the Phenylalanine side chain is removed from its lipophilic binding pocket, FIG. 2B. As a result of this conformational change, new binding sites just adjacent to the “Gatekeeper Region” become accessible. Therefore, compounds that bridge from the HGM to these new “Selectivity Sites” can build in new structural elements that take advantage of differences between kinases in this region. Because the DFG-out conformation represents a minor population, Type-II inhibitors display time dependent kinetics resulting from slow on rates and the energetics of conformational equilibration. Consequently, potent Type-II inhibitors must also exhibit slow rates of disassociation while further kinetic advantages of Type-II result from this conformations inability to bind ATP and be recognized by upstream regulatory kinases. (Goshe A. K. J. Med. Chem. (2008), 51(17):5149-5171).
Typically, Type-II inhibitors incorporate a carboxamide, urea, or similar H-bond bridging bioisoster linkage to span from the Selectivity Sites into the ATP binding region. Therefore, Type-II inhibitors can be viewed as a Hinge-Gatekeeper Motif (HGM) appropriately connected to a lipophilic template that penetrates into and is complimentary to the adjacent “Selectivity Sites”. Using this construct, Type-I inhibitors have been converted to Type-II inhibitors (Liu Y., Nature Chemical Biology (2006) 2:358-364; Kufareva I., J, Med. Chem. (2008), 51(24):7921-32).
Type-III inhibitors are relatively uncommon as they occupy a region adjacent to but not overlapping with the ATP binding site that does not require significant conformational change of the DFG-loop. Because Type-III inhibitors coexist with ATP binding, they are non-competitive with ATP and unaffected by the high physiological ATP concentrations. Since Type-Ill inhibition is rarely observed, inhibitors of this type offer potential selectivity advantages over Type-I inhibitors. MEK1 and MEK2 are important kinases for which Type-III inhibitors have been reported (Tecle H., Bioorg. Med. Chem. Lett. (2009), (19)1: 226-229).
Type IV inhibitors compete with a protein kinase substrates and target regions outside the ATP binding site that may avoid some crucial problems associated with the more conventional ATP competitive kinase inhibitors, such as the development of drug resistance as a result of accumulating mutations in the ATP binding site of the kinase.
Type V inhibitors are defined as a family of allosteric inhibitors that recognizes a binding domain well outside the ATP-binding cleft and not necessarily close to the substrate pocket. Therefore, this type of inhibition can be very specific for a given kinase.
Use of 5-Membered Heterocyclic Scaffolds at the ATP Binding Site:
The tri-substituted imidazole template has been applied very successfully to the Type-I inhibition of kinase. (Takle H., Bioorg. Med. Chem. Lett. (2009), (19)1: 226-229.) and knowledge gained from binding at the ATP site (Bennett et. al., WO2007105058A2) has been extended by appending functionality to provide structurally related Type-II inhibitors. (Tang J., Bioorg. Med. Chem. Letts., (2008), 18:4610-4614), FIGS. 3 A & B. In fact, structural information gained from Type-I inhibitors has been translated to the design of a type-II inhibitor intentionally by the same group (Wolin R. L., Bioorg Med Chem Letts. (2008), 18(9):2825-2829), FIGS. 3 C & D. These examples illustrate how chemotypes that target the ATP binding site can be adapted to provide Type-II inhibitors through an appropriate urea, amide, or ether, FIGS. 3 E & F, linkage to an additional lipophilic aromatic ring that occupies the lower selectivity-site (Meyers M. J., Bioorg Med. Chem. Letts. (2010) 20:1543-1547). It is estimated that approximately 50 of the 518 kinases adopt the DFG-out conformation (Fabian M. A., Nat Biotechnol (2005) 23:329-336), thus limiting the targets for Type-II inhibitors. However, recent studies suggest that the DFG-out conformation may be more common than initially thought (Kufareva I, J, Med. Chem. (2008), 51(24):7921-32).
Use of Urea-Linked 5-Membered Heterocyclic Scaffolds in Type-II Inhibitors:
Urea linked aryl-substituted 5-membered heteroaryl scaffolds have been used previously to create favorable “Selectivity Site” interactions for the Type-II inhibition of kinases, FIG. 4 A-D, (Smith R. A., Bioorg. Med. Chem. Letts. (2001), 11: 2775-2778; Regan J., J. Med. Chem. (2002), 45: 2994-3008. Regan J., J. Med. Chem. (2003), 46: 4676-4686; Michellotti E. L., et. al., WO/2006/062982; Raeppel. S, Bioorg. Med. Chem. Letts. (2009), 19:1323-1328). The urea function acts as a critical hydrogen bonding bridge between a conserved Glutamate side chain and the aspartyl NH from the DFG-loop. In addition to urea linages, carboxamide linages have also been widely employed to profide Type-II inhibitors, (Zhang J, Nat. Rev. Cancer (2009) 9(1): 28-39). The closest prior art to that described herein is the cyclic urea c-Met inhibitor illustrated in FIG. 4-D. To our knowledge, the only previous description of aType-II kinase inhibitor containing an ortho-Aryl-substituted 5-membered heteroaryl carboxamide scaffold is from our previous work, (Dietrich J., Bioorg. Med. Chem. (2010), 18(1): 292-304), which is limited to the imidazole scaffold with a quinazolinone HGM, and is the only example of a carboxamide linked aryl-substituted 5-membered heteroaryl Type-II inhibitor. We have recently become aware of a recent patent (Son, J B, et. al., WO 2011093684) which claims the use of 5-arylmethyl-2-methyl-pyrazole-4-carboxamide kinase inhibitors, see Table 1, HGM#10. In this patent, the lack of direct aryl substitution on the pyrazole ring significantly changes the scaffold geometry that is necessary for the unique properties describe for the scaffolds herein.
Compared to the limited structural variations that have been utilized to interact at the “Selectivity Site” of the DFG-out conformation of kinases, the structural Hinge Gatekeeper-interacting Motif (HGM) variations that bind to the ATP binding site has been well studies. A survey of the kinase inhibitor are reveals this diversity, see Tables 1-9. In these tables, the HGM amine is illustrated in the first column while the amine capping group is illustrated, where applicable, in the adjacent column. Variations on the imatinib HGM are depicted in Table 1, while a series of difluoroanaline HGMs normally capped by a sulfonyl group are depicted in Table 2. A series of biaryl HGM amines are summarized in Table 3 and a series of HGMs with Bicyclic Gatekeeper Interacting Ring Systems are illustrated in Table 4. Following Tables 5 and 6, which illustrate the wide variety of diaryl ether HGMs that have been explored, Table 7 displays related but atypical linker strategies between the Hinge and Gatekeeper-interacting groups. Table 8 illustrates how derivatives of heteroaryl linked ATP site inhibitors can be adapted to the design of Type-II inhibitors. Lastly, Table 9 illustrated non-amine HGMs that, although not directly applicable to the construction of Type-II inhibitors, could be adapted, as indicated, for construction of Type-II inhibitors.
TABLE 1Hinge Gatekeeper-interacting Motifs (HGMs) with 4-methyl-1-3Disubstituted-Phenyl Amine Gatekeeper-interacting Groups.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group1 2 3 4 5 6 7 8 9 10Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure1ImatinibABLZimmerman 19961IEP.pdbKDR, Kit,Zimmermann, J. et al. Bioorg.Imatinib in ABLPDGFR,Med. Chem. Lett. 1996, 6,CSF-1R,1221-1226. Order# A2308FLT-3, DDRsales@tciamerica.com2Zhang 2010WO2010-US418723MasitinibKitA01.743.786(AB1010)PDGFRwww.aurorafinechemicals.comFGFR34PonatinibBCR-ABL,Huang 20103CS9.pdbmutants,Huang, WS et al. Journal ofPoltinib in ABLKDR, FGFR1,Medical Chemistry 2010, 53PDGFRα,(12): 4701-19FLT3, LYN5GSK Cpd-CSF-1RBaldwin 2008Best CSF-1R14dLCK, EGFR,Baldwin I., etal. Bioorg. Med.from libratryErbB4, KDRChem. Lett. 18 (2008) 5285-approach52896BafetinibABL LYNKamitsuji 2007(INNO-406)Kamitsuji Y, et al. BioorgMed Chem Lett. (2007) 17:12-177AZ628B-RAFAquila 2007PDGFR-a/bAquila, B., Lyne, P., Pontz, T.:KIT, CSF1RWO2007113558 (2007)8TIE-2Hodous 2007Hodous B. L., et.al. Bioorg.Med. Chem. Let. (2007) 17:2886-18899ZM 336372CRAFHall-Jackson 1999Hall-Jackson C. A., et.al.Chem Biol (1999) 6(8): 559-68101318242-17-Son 20119PSon, J B, et.al.,WO 2011093684
TABLE 2Representative 4-6-Difluoro-1-3-disubstituted Gatekeeper ContainingInhibitors.ATP-Site InteratingReference orComment orHGMGroup-Hinge-InhibitorKinasesVendorCrystal#Gatekeeper Motif-HGMSulfonamide CapName or ID#InhibitedSourceStructure11B-RAFIgnacuo 2011 Ignacio A., et.al. WO 2011025940Novel Type-I Inhibitor 12vemurafenib (Zelboraf) PLX-4032B-RAF (V600E) 31 nM C-RAF (48 nM) SRMS (18 nM) MAP4K5 51 nM FGR (63 nM) B-RAF 100 nMBollag 2010 Bollag, G., et.al., Nature (2010) 467: 596-5993OG7_B.pdb Raf (V600E) 13PLX-4720B-RAFTsai 2008 Tsai, J., et.al., Proc. Natl. Acad. Sci. Usa (2008) 105: 3041-30463C4C.pdb B-RAF 14PLX-3203B-RAFTsai 2008 Tsai, J., et.al., Proc. Natl. Acad. Sci. Usa (2008) 105: 3041-30463C4D.pdb BRAF 15B-RAFWenglowsky 2011 Wenglowsky, S., et.al. ACS Medicinal Chemistry Letters (2011) 2: 342-3473TV4.pdb BRAF 16B-RAFWenglowsky 2011 Wenglowsky S., et.al., Bioorg. Med. Chem. Lett. (2011) 21: 5533-55373TV6.pdb BRAF 17B-RAFJoachim 2011 Joachim R., et.al., WO(2011) 025951 A1Novel Type-I Inhibitor 18Wenglowsky 2011 Wenglowsky S., et.al., Bioorg. Med. Chem. Lett. (2012) 22: 912-915Novel Type-I Inhibitor 19B-RAF VEGFR-2Ren 2012 Ren, L., et.al. Bioorg. Med. Chem. Lett (2012). 22: 3387-33914E4X.pdb B-Raf
TABLE 3Representative Biaryl-amine Hinge Gatekeeper-Interacting Motifs (HGMs).ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group20 21 22 23 24 25 26Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure20AC-220FLT-3WO/2005/048953;WO/2009/03875721BRAFBerger 20093II5.pdbBerger, D. M., et.al.BRAFBioorg. Med. Chem. Lett.(2009) 19: 6519-6523US2007021918622PF-4594755PYK2Shena 2011Shena C. J., et.al.,Experimental Cell Research(2011) 317: 1860-187123LinifanibPDGFR-bShankar 2007(ABT-869)CSF-1RD. B. Shankar, etal. BLOOD375.4KDR109(8), 2007, 3400-3408WO/2004/11330424KDR, TIE-2Dai 2008Dai Y., et.al., Bioorg. Med.Chem. Lett. (2008) 18: 386-39025KDRDai 20085Dai Y., et.al., J. Med. Chem.(2005) 48: 6066-608326KDR, TIE-2Miyazaki 2005Miyazaki Y., et.al., Bioorg.Med. Chem. Lett. (2005)15: 2203-2207.
TABLE 4Representative HGMs with Bicyclic Gatekeeper Interacting Ring Systems.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group27 28 29 30 31 32 33Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure27Chen 2007Chen, Ning; Hu, EssaWO 2007-US1638328BRAFSmith 20093IDP.pdbSmith, A. L., et.al.,BRAFJ. Med. Chem. (2009) 52:6189-619229Raf-265RAFAmiri P, et al.(CHIR-265)VEGFRUS20070299039Mol Cancer Ther 2010; 9: 358-36830Raf265RAFAmiri P, et al.des-methylVEGFRUS20070299039derivative31RAFUS 2003-675927VEGFRWO 2003-US1011732KDRHasegawa 2007Hasegawa, M., et.al.,J. Med. Chem (2007) 50: 4453-447033Bauer, D., et.al., Bioorg.Med. Chem. Lett. (2008)18: 4844-4848
TABLE 5Representative Diaryl Ether Hinge Gatekeeper-Interacting Motifs HGMs.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteInhibitorHGM #Gatekeeper Motif-HGMInteracting GroupName or ID#34BIRB 796 Dorama- pimod 35Sorafenib Nexavar 36Regorafenib BAY 73-4506 37 38 39 40 41Comment orKinasesCrystalHGM #InhibitedReference or Vendor SourceStructure34p-38Regan 2003IKV2.pdbRegan, J., et.al. J. Med.Human p38Chem. (2003) 23; 46(22): 4676-MAP Kinase in86.Complex withBIRB 79635KDR, Kit,Wood 1998PDGFRs,Reidl B., et.al.,CSF-1R,WO-1998-53559FLT-3,DDR, Raf,Tie236VEGFR-2/3,Onyx-Sorafenib RET, KIT,U.S. Pat. No. 7,351,834 (filed on 1999)PDGFR, andRafs37B-RafNiculescu-Duvaz 2009Niculescu-Duvaz, D., et.al.,J. Med. Chem. (2009) 52:2255-226438B-RafMenard 2009Menard, D., et.al.,J. Med. Chem. (2009)52: 3881-389139B-RAFOkaniwa 20124DBN.pdbVEGFR-2Okaniwa, M., et.al.,BRAFJ. Med. Chem (2012) 55: 3452-347840KDR TIE-2Hasegawa 2007Hasegawa, M., et.al.,J. Med. Chem (2007) 50: 4453-447041B-RAF (V600E)Whittaker 2010BRAF, CRAF,Whittaker S., et.al. CancerSRC, LCK,Res; (2010) 70(20) 8036-PDGFR-a, p38-8044.a, p38-g,
TABLE 6Representative Diaryl Ether Hinge Gatekeeper-Interacting Motifs HGMscontinued.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group42 43 44 45 46 47 48 49Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure42KI-20227CSF-1R >Ohno 2008R and S isomers480.54KDR > Kit,Ohno, H. et al. Eur. J.are slightlyPDGFR-bImmunol. (2008) 38: 283-291.different43BMS 777607c-METSchroeder_2009 Schroeder3F82.pdbGM, et.al. J. Med. Chem.(2009) 52(5): 1251-1254.44c-METSchroeder 20093CE3.pdbSchroeder G. M., et.al.J. Med. Chem. 52(5), 2009,1251-445c-METSchroeder_2009 Schroeder3CTH.pdbGM, et.al. J. Med. Chem.(2009) 52(5): 1251-1254.46Foretinibc-METQian 2009GSK1363089VEGFR-1,Qian F., et.al., Cancer ResXL880VEGFR-2(2009) 69: 8009-8016EXEL-288047MGCD265c-METU.S. Pat. No.VEGFR-1,7,772,247VEGFR-2,VEGFR-3,RON andTIE248LenvatinibVEGFR 2/3Matsui 2008(E7080)Matsui, J.; et.al., ClinicalCancer Research (2008) 14(17): 5459-6549PYP-4-0001Scientific Laboratory Inc.sales@sphinxscientificlab.com
TABLE 7Hinge Gatekeeper-Interacting Motifs (HGMs) with Atypical Linker Groups.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group50 51 52 53 54 55 56 57 58Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure50AstraCSF-1R>D. A. Scott etal. Bioorg Med.ZenecaEphA2, Hck,Chem. 198(2008) 4794-4797Cpd-27Fyn, cRaf,WO/2007/071955KDR, SrcPDGFRs51KIT CSF1RUS20070032519WO/2007/01389652p38Milian 20112YIX.pdbMillan, D. S., et.al.,p38 andJ. Med. Chem. (2011) 54: 7797trialopyridineinhibitor53PF-4618433PYK2Han 20093FZT[.pdb PYK2Han, S., et.al., J. Biol. Chem.and PF-4618433(2009) 284: 13193-1320154Cytopia-CSF-1RC. J. Burns etal. Bioorg. Med.StereoAustraliaKit, DDR1Chem. Lett. 19 (2009) 1206-Chemistry441.48PDGFRs,1209ImportantVEGFR-1,2,3FRK, RET55TelatinibPDGFR-bNeeltje 2009(BAY 57-VEGFR-2/3Neeltje Steeghs N., J Clin9352)KITOncol (2009) 27: 4169-417656RAFBartkovitz D. J., et al.,US2007006060757MK-2461c-MetPan B-S., et.al., Cancer ResRon, Flt1-4,2010; 70(4), 1524-1533PDGFRβ,FGFR1-3KDR, TrkA/Band Mer58PYP-4-0025Scientific Laboratory Inc.sales@sphinxscientificlab.com
TABLE 8Hinge Gatekeeper-Interacting Motifs (HGMs) with Azole Linking RingSystems.ATP-Site InteratingGroup-Hinge-Type-II-Selectivity SiteHGM #Gatekeeper Motif-HGMInteracting Group59 60 61 62 63 64Comment orInhibitorKinasesCrystalHGM #Name or ID#InhibitedReference or Vendor SourceStructure59DabrafenibBRAFStellwagen J. C., et.al. Bioorg.GSK2118436AMed. Chem. Lett. (2011) 21:4436-4440WO201104441WO2011044414WO201104723860BRAFTang 2008Tang J., et.al., Bioorg. Med.Chem. Lett. (2008) 18: 4610-461461TIE-2Lee 2010Lee J., et.al., Bioorg. Med.Chem. Lett. (2010) 20:1573-157762TIE-2Adjabeng G., etal.WO 2009076140 A163B-RAFStellwagen J. C. etal.,Bioorg. Med. Chem. Lett.(2011) 21: 4436-4440WO2009032667 A164BKM120Pan-PI3KBurger 2011NVP-Burger T. M., et.al., ACSBKM120Med. Chem. Lett. (2011)2: 774-779
The structures in Table 9 represent kinase inhibitors that contain interesting HGMs that, because they lack an appropriately positioned amine group, can not be directly used to construct Type-II inhibitors. As illustrated in FIG. 3, ATP binding site inhibitor motifs from Type-I inhibitors have been adapted to the construction of Type-II inhibitors (Wolin R. L., Bioorg. Med. Chem. (2010), 18(1): 292-304; Meyers M. J., Bioorg Med. Chem. Letts. (2010) 20:1543-1547).
TABLE 9Type-I Inhibitors that could be adapted to Library HGM Amines as Indicated.Type-II-Select-ivitySiteReferenceCommentATP-Site InteratingInter-InhibitorororHGMGroup-Hinge-actingName orKinasesVendorCrystal#Gatekeeper Motif-HGMGroupID #InhibitedSourceStructure65Crizotinib PF02341066ALK 66None- Type-IDasatinibABL KDR, Kit, PDGFR, CSF-1R, FLT-3, DDRLombardo, L. J. et al. J. Med. Chem. 2004, 47, 6658- 6661.2GQG.pdb Dasatinib in ABL 67Want Reverse AmideGeuns-Meyer, S. D.; et. al., WO 2005113494 A2 68Want Reverse AmidePCT Int. Appl., 2005113494, 01 Dec. 2005 69Replace Benzyl- oxy Group with Amine FunctionPfizer-12b 448.47CSF-1RMeyers 2010 Meyers M. J., et al. Bioorg. Med. Chem. Lett. (2010) 20:1543-15473LCO.pdb CSF1R 70Replace Meth- oxy Group with Amine FunctionPl3K/ mTORLiu 2010 Liu, K. K.-C., et. al., Bioorg. Med. Chem. Lett. 2010, 20 (20), 6096- 6099.3ML9.pdb Pl3K- gamma 71Replace Hydroxy Group with Amine FunctionWO 2007- 123892 72None- Type-I Add Amine Functions as IndiacatedPD173955ABL KIT, SRC, HCK, LCK,Klutchko 1998 Klutchko, S. R. et al. J. Med. Chem. 1998, 41, 3276- 3292.1M52.pdb Abl 73Pelitinib (EKB- 569)(EGFR) ErbB-1, −2 and −4J. Med. Chem. 2003;46, 49-63Covalent Inhibitor
An efficient synthesis of 2-trifluoromethyl-4-aryl-imidazole-5-carboxylic acids FIG. 5A, has been reported (Hagiwara K., et. al, WO/1995/004724). Quinazolinone containing amides, FIG. 5B, have been claimed to potently inhibit the oncongenic B-Raf (V600E) mutant kinase (Aquila B., et. al, WO2006/024834). Attachment of a 2-trifluoromethyl-4-aryl-imidazole-5-carboxylic acid, A, to this quinazolinone containing (HGM) resulted in a homologous series of hybrid compounds, FIG. 5C, some of which were reported to be highly potent B-Raf(V600E) inhibitors (Dietrich J., et al. Bioorg. Med. Chem. (2010), 18(1): 292-304).
This previous disclosure described computational experiments using 1UWJ.pdb, the co-crystal structure of B-Raf(V600E) with Sorafenib, in which no attractive low-energy pose were produced during docking studies. This poor fit, thought to result from a steric clash between the inhibitor and Glutamate 500 in 1UWJ.pdb, FIG. 6A, prompted exploration of alternate binding modes. Thus, this report proposed an alternative mode of binding based on docking experiments using the DFG-out monomer-B co-crystal structure of B-Raf(V600E) with PLX-4720 (Tsai J., PNAS USA. (2008) 105(8):3041-6). By utilizing this co-crystal structure, 3C4C.pdb, the proposed negative steric interaction with glutamate 500 appeared to be removed, FIG. 6B.
FIG. 6 illustrates the binding mode of quinazolinone containing inhibitors with B-Raf.
This publication also described the structure activity relationship within a small series of structurally related compound which all shared the quinazolinone HGM and imidazole scaffold. The only structural variations reported were limited to the 2- and 5-positions of an imidazole scaffold with the three compounds depicted in FIG. 7 displaying the most potent activity against B-Raf(V600E). The apparent time dependence reported was consistent with Type-II inhibition. The phosphor-protein gel assay used in this report revealed sub-nanomolar IC50 values for these three inhibitors when the inhibitor and enzyme were preincubated for 1 hour prior to addition of the MEK1 substrate. When evaluated against a panel of 96 kinases, Table-10, all three compounds displayed potent and selective inhibition of B-Raf(V600E), B-Raf(Wt), C-RAF(Raf-1), PDGFR-α, PDGFR-β, c-Kit, and p38-α.
TABLE 10Summary of inhibition profiles for the imidazole quinazolinone inhibitorsKIN-035, KIN-038, and KIN-057, (Dietrich, 2010).InhibitorCodeKIN-035KIN-038KIN-057 Ambit Biosciences Gene SymbolABL1ABL1(E255K)Inhibition ScaleABL1(T315I)   X > 25% @ 1 μMACVR1B   XX > 50% @ 1 μMADCK3   XXX > 75% @ 1 μMAKT1XXXX > 90% @ 1 μMAKT2ALKAURKAAURKBAXLBMPR2XBRAFXXXXXXXXXXBRAF(V600E)XXXXXXXXXXXBTKXCDK11XXXXXCDK2CDK3XCDK7XCDK9CHEK1CSF1RXXXXCSNK1DCSNK1G2DCAMKL1XXXDYRK1BEGFRXEGFR(L858R)XEPHA2XXERBB2XXERBB4XERK1FAKFGFR2FGFR3XFLT3GSK3BIGF1RIKK-alphaXIKK-betaXINSRJAK2X(catalytic)JAK3(catalytic)JNK1JNK2JNK3KITXXXXXXXXXXXKIT(D816V)XLKB1MAP3K4Inhibition ScaleMAPKAPK2   X > 25% @ 1 μMMARK3   XX > 50% @ 1 μMMEK1   XXX > 75% @ 1 μMMEK2XXXX > 90% @ 1 μMMETMKNK1XXXMKNK2XMLK1p38-alphaXXXXXXXXXp38-betaXXXPAK1PAK2PAK4PCTK1XPDGFRAXXXXXPDGFRBXXXXXXXXPDPK1PIK3C2BPIK3CAXXPIK3CGPIM1PIM2PIM3PKAC-alphaPLK1PLK3PLK4XXXPRKCEXXXRAF1XXXXXXXXXXXXRETRIOK2XXROCK2RPS6KA3(Kin.)SNARKSRCSRPK3TGFBR1TIE2TRKATSSK1BTYK2XX(catalytic)ULK2XXVEGFR2XYANK3ZAP70Compounds were assayed at 1 μM concentrations in duplicate without preincubation.An X-indicates better than 25% inhibition, XX-indicates better than 50% inhibition, XXX-represents better than 75% and XXXX represents better than 90% inhibition under the conditions of these assays.Noteatypical Structure-Activity Relationship of KIN-035 for CSF1R compared to KIN-38 and KIN-57.
The present disclosure addresses and interconnects two important applications relating to the treatment of diseases. Because understanding of the disease specific roles, complex interactions, mechanisms of dysregulation, activating mutations, and compensatory back-up systems of kinase pathways, i.e. “Systems Biology”, continues to evolve, multi-targeted kinase (MTK) inhibitors with unique properties and selectivity profiles will continue to be needed. The development of advanced tools for the non-invasive mechanism-based characterization and monitoring of disease in preclinical, clinical, and therapeutic settings is also perceived as a critical unmet need.
The present disclosure describes a novel scaffold geometry and its application to the design and preparation of selective or multi-targeted kinase (MTK) inhibitors as therapeutic agents and/or disease specific PET imaging agents. Enabling technologies for the early diagnosis, accurate characterization, patient specific treatment, and real time monitoring of therapies will be essential for the realization of personalized medicine. By combining therapeutic and imaging agents that share similar structural elements and/or activity profiles, significant synergies can be realized in clinical development and personalized medicine. Taken together, these concepts constitute a platform technology with unique applications and utilities. Although this disclosure focuses on representative examples for the purposes of illustration, the implications of and applications for this platform technology are quite broad and, using the information disclosed herein, one skilled in the construction of Type-I and/or Type-II kinase inhibitors and the mode of binding of Hinge-Gatekeeper interacting Motifs (HGM) should be able to easily employ this platform technology.